In vitro synthesis of vitamin D-3 by cultured human keratinocytes and fibroblasts: action spectrum and effect of AY-9944

With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skin's role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting.     

A preliminary note on the use of milk substitutes in the early weaning of dromedary camels

The experiment was performed using two young male camels which weighed 24 and 36 kg respectively at birth. Each young camel was weighed and abruptly separated from its mother after 30 days of nursing (or at 1 month of age). The weaned calves were fed milk substitutes prepared commercially for lambs by Mabarot Chemical and Veterinary Products (Israel). The weanling camels averaged daily weight gains of 0.400 and 1.0 kg per day respectively during the 30 day initial period when the milk substitutes were used. Following the period when milk substitutes were used, the camels achieved normal growth to arrive at 135 and 145 kg respectively at 6 months of age.     

Minireview: Metabolic control of the reproductive physiology: Insights from genetic mouse models

This article is part of a Special Issue Energy Balance.     

Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase

The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or dATP) is not required to initiate or sustain highly processive synthesis. The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.     

Behavioral interactions between three birch aphid species and Adalia bipunctata larvae

First and fourth instars of three birch aphid species were exposed to first and fourth instars of Adalia bipunctata (L.), the most common aphid predator on silver birch, Betula pendula Roth, in northern California. Defensive behavior differed by aphid species. Euceraphis betulae (Koch) (Eb), the most successful escapee, was highly mobile and frequently walked away from coccinellid larvae. Betulaphis brevipilosa Börner (Bb), a flat, sessile species, was the least successful aphid at actively escaping from A. bipunctata larvae, but could passively escape detection when coccinellid larvae walked over nymphs and did not perceive them. Active escape behavior was much safer for aphids than passive avoidance of detection. Both instars of Eb and fourth instars of Callipterinella calliptera (Hartig) (Cc) escaped from coccinellid larvae more frequently when approached from the front, apparently using vision for pre-contact detection of A. bipunctata. These aphids avoided physical contact with larger predators more often than with smaller predators. Level of predation by A. bipunctata on these three aphid species is dependent upon types of aphid defense.

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Résumé Des larves de premier et troisième stades de 3 espèces de pucerons du bouleau: Betulaphis brevipilosa, Callipterinella calliptera et Euceraphis betulae ont été exposées aux attaques des larves du premier et du quatrième stades d'Adalia bipunctata, prédateur le plus fréquent de pucerons sur Betula pendula en Californie du Nord. Le comportement défensif dépend beaucoup de l'espèce de puceron. E. betulae, qui s'échappe avec le plus de succès, est très mobile et s'écarte fréquemment des larves agressives de coccinelles. B. brevipilosa, espèce plate, sessile, a le moins de succès dans la protection active contre les attaques des larves de A. bipunctata, mais elle peut échapper passivement à la détection des larves de coccinelles qui ne peuvent pas les discerner lorsqu'elles circulent parmi les larves de pucerons. E. betulae (aux deux stades) et C. calliptera (au quatrième) échappent le plus aux larves de coccinelles qui attaquent de front, ce qui laisse supposer que la vision permet de détecter A. bipunctata avant le contact. Le succès des larves de coccinelles varie selon les stades du puceron et de la coccinelle. Les possibilités d'obtention de niveaux de prédation élevés de ces 3 pucerons par A. bipunctata dépend du type de protection du puceron.

     

Interferon effects upon fluorouracil metabolism by HL-60 cells

In order to better understand the synergistic antiproliferative effects of interferon in combination with fluorouracil (FUra), we studied effects of alpha 2-interferon upon FUra induced inhibition of thymidylate synthase of HL-60 cells. The 50% inhibitory dose for FUra decreased from approximately 75 microM to 10 microM following interferon treatment, as measured by whole cell activity assays. Enhanced FUra inhibition of cytosolic [3H] - FdUMP binding of interferon treated cells was also noted. FdUMP accumulation following FUra treatment increased over 10 fold in interferon treated cells, but dUMP did not increase. These results suggest that interferon can sensitize cells to FUra inhibition of thymidylate synthase by enhancing accumulation of FdUMP.     

Stimulation by tumor necrosis factor of HL-60 thymidine salvage pathway metabolism dissociated from proliferation

The effect of human tumor necrosis factor (TNF) on early-passage HL-60 cells was studied. A transient phase of increased [3H]thymidine (TdR) incorporation was noted at 20-24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S-phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [3H]TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [3H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL-1, IL-2, IL-3, IL-4, G-CSF, M-CSF, GM-CSF or alpha- or gamma-interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [3H]TdR incorporation. mRNA synthesis inhibition with alpha-amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [3H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S-phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.